Blotting techniques such as Southern blotting, Northern blotting, and Western blotting allow for the transfer and detection of DNA, RNA, and proteins, respectively. Southern blotting involves separating DNA fragments via gel electrophoresis, transferring them to a membrane, and using a probe for hybridization to detect specific DNA sequences. Northern blotting detects RNA transcripts by separating RNA on a gel, transferring it to a membrane, and labeling it with a probe. Western blotting identifies proteins by separating them on an SDS-PAGE gel, transferring them to a membrane, and using primary and secondary antibodies to detect specific proteins based on antigen-antibody binding. These techniques have various applications in research, diagnostics, and forensics

2. BLOTTING
TECHNIQUES

3. What is blotting?
Technique for transferring
• DNA
• RNA
• Proteins
onto a carrier so they can be separated,
and often follows the use of a gel
electrophoresis.

4. TYPES OF BLOTTING
TECHNIQUES
BLOTTING
TECHNIQUES
Southern Blot
It is used to detect
the DNA.
Northern blot
It is used to detect
the RNA.
Western blot
It is used to detect
proteins.

5. Blotting sheet
• Whatman 3mm paper….. world’s most widely used
blotting paper.
 REASON????
• high quality
• purity
• consistency

6. Creating the Sandwich
• The sandwich consists of :
filter paper
Nitrocellulose membrane
gel matrix
another piece of filter paper

7. SOUTHERN BLOTTING
History:
• Named after Sir Edwin
Southern
• Developed in 1975

8. SOUTHERN BLOTTING
“Used to detect the DNA”
• This method Involves:
 Separation
 Transfer
 Hybridization.
• This DNA can be:
• Single gene
• Part of a larger piece of DNA……..viral genome
“The key to this method is Hybridization”

9. Hybridization
“Process of forming a
dsDNA molecule between a
ssDNA probe and a ss-target
patient DNA”

10. PRINCIPLE
 The mixture of molecules is separated.
 Immobilized on a matrix.
 Probe addition to the matrix to bind to the
molecules.
 Unbound probes are removed.
“The place where the probe is connected
corresponds to the location of the immobilized
target molecule.”

11. Steps in southern blotting
The DNA is digested
Fragments
Gel electrophoresis
Transfer to membrane
Probing
Autoradiogram

13. APPLICATIONS
Southern blotting is used in:
• Gene discovery
• Mapping
• Evolution
• Development studies
• Diagnostics
• Forensics

14. Northern Blotting
History:
Northern blotting was developed by James
Alwine and George Stark at Stanford
University.
Northern blotting is a technique for
detection of specific RNA sequences

15. Steps involved in N.B
RNA isolation
Loading of sample on Agarose gel
Blotting on nitrocellulose membrane
Labeling with probe
Washing to remove unbound probe
Detection by autoradiogram

17. APPLICATIONS
• A standard for the direct study of gene expression at
the level of mRNA (mRNA transcripts)
• Detection of mRNA transcript size
• Study RNA degradation
• Study RNA splicing - can detect alternatively spliced
transcripts
• Study RNA half-life
• Study IRES (internal ribosomal entry site) – to
remove possibility of RNA digestion vs. 2nd cistron
translation.

18. Western blotting
Discovery???
• Dr. Douglas Lake of the University of Arizona School of
Medicine's Department of Microbiology and Immunology
“A technique in which proteins are
separated by gel electrophoresis and
transferred to a membrane sheet. A
specific protein is then identified through
its reaction with a labeled antibody.”

19. Principle
This technique works on
the principle on
“Antigen-Antibody”
relationship

20. Prerequisite for W.B
The SDS PAGE technique is a
prerequisite for Western
blotting.
“SDS (sodium dodecyl sulfate) is a
detergent (soap) that can dissolve
hydrophobic molecules but also has
a negative charge (sulfate) attached
to it.”

21. Steps in W.B
1. Gel electrophoresis:
The proteins are separated according to size.
2. Membrane Transfer:
Transferring to nitrocellulose by applying current.
3. Blocking:
Done to prevent non-specific protein interactions
between the membrane and the antibody protein.

23. Blocking in W.B
• The blot is incubated with a generic protein (such as
milk proteins) which binds to any remaining sticky
places on the nitrocellulose.
• An antibody that is specific for the protein of
interest(the primary antibody - Ab1) is added to
the nitrocellulose sheet and reacts with the antigen.
• Only the band containing the protein of interest
binds the antibody, forming a layer of antibody
molecules .

24. Cont..
• Following several rinses for removal of non-
specifically bound Ab1, the Ab1-antigen complex on
the nitrocellulose sheet is incubated with a second
antibody (Ab2), which recognizes the primary
antibody and binds it.
• Ab2 is radioactively labeled, or is covalently linked to
a reporter enzyme, which allows to visualize the
protein-Ab1-Ab2 complex

25. Applications
• To identify the specific proteins
• To identify their masses
• The confirmatory HIV test to detect anti-HIV antibody in a
human serum sample.
• The definitive test for Bovine spongiform encephalopathy
(BSE, commonly referred to as 'mad cow disease').
• Some forms of Lyme disease testing also employ Western
blotting.

27. Protein gel (SDS-PAGE) that has
been stained with Coomassie Blue.

28. Faster Way to Western
Blot
• The Quick Western Kit provides a universal detection
reagent that can be combined with the primary
antibody incubation step, eliminating the need for a
secondary antibody incubation step.
• This kit works with a variety of primary antibodies.
• This reduces the overall time to complete a Western
blot.

29. Standard Western Blot